Our pre-clinical studies showed that 13-cis-retinoic acid (13-cis-RA) can inhibit cell growth and decrease expression of the MYCN oncogene in neuroblastoma (NBL) cells in vitro. These data led us to conduct a phase I and then a randomized phase III trial which showed that high-dose, pulse 13-cis-RA significantly improved survival for high-risk NBL when given after intensive chemotherapy. Not all patients benefited from 13-cis-RA, and we found that some NBL cell lines derived from patients who were clinically resistant to 13-cis-RA are resistant to 13-cis-RA and trans-RA in vitro. We discovered that the RA derivative N-(4-hydroxyphenyl) retinamide (fenretinide; 4-HPR) is highly cytotoxic for NBL cell lines that are resistant to 13-cis-RA. The hypothesis of this proposal is that 4-HPR will further improve the survival of high-risk NBL patients by eradicating tumor cells, including those cells that are resistant to 13-cis-RA. This project will explore the molecular mechanisms by which 4-HPR selectively induces tumor cell death. We showed that 4-HPR caused large increases in the levels of ceramide (a lipid involved in apoptotic signaling) in NBL cell lines and that (like exogenous ceramide) 4-HPR cytotoxicity involved both apoptosis and necrosis. We will continue this work by determining if ceramide synthetic enzymes are induced by 4-HPR or if ceramide metabolic enzymes are down-regulated. To determine if cytotoxicity mediated by high-dose 4-HPR is due to increased ceramide we will transfect a NBL cell line with an inducible vector containing glucosylceramide synthase, which will shunt ceramide in 4-HPR treated cells to a non-toxic form. Cell lines will be identified and developed that are resistant to 4-HPR to determine if 4-HPR-resistance is mediated by a failure to increase ceramide in response to 4-HPR or by "shunting" the ceramide to non-toxic forms. Agents that inhibit ceramide shunting will be evaluated for their ability to enhance the cytotoxicity of 4-HPR for NBL cell lines. Combinations of agents with 4-HPR that have enhanced cytotoxicity against NBL cell lines in vitro will be tested for general toxicity against fibroblasts and myeloid progenitors. and for toxicity and efficacy against NBL xenografts. 4-HPR and enhancing agents will be evaluated for their efficacy against NBL cell lines that are resistant to 13-cis-RA and chemotherapeutic drugs. This project, in combination with our ongoing phase I trial of 4-HPR, will provide data to optimize clinical trial design employing 4-HPR against NBL. We anticipate that data generated will support developing a future clinical trial testing the ability of 4-HPR to improve outcome for high risk NBL when given after myeloablative therapy and 13-cis-RA. Elucidating mechanisms of action for 4-HPR should allow us to identify clinically useful agents that will enhance the anti-cancer activity 4-HPR for testing in future clinical trials.